Method for detoxification of mycotoxins

ABSTRACT

Disclosed is a method for detoxification of mycotoxins wherein mycotoxin is contacted with a glucosyltransferase in the presence of an activated glucose.

The present invention relates to a method for detoxification ofmycotoxins as well as a method for producing derivatives of mycotoxins.

A complex of closely related species of the genus Fusarium isresponsible for destructive and economically very important diseases ofcereal crops (Fusarium head blight, FHB, of wheat and barley) and maize(Fusarium ear rot). In years with climatic conditions that favor thedevelopment of the fungi, Fusarium infections can reach epidemicproportions (1). Diseases caused by the plant pathogens do not onlyseverely reduce yield, but also result in contamination of grain withunacceptable high amounts of mycotoxins, a problem of world-widesignificance. A toxin class of particular concern to human and animalhealth are trichothecenes, sesquiterpenoid epoxides which are potentinhibitors of eukaryotic protein synthesis. More than 180 compounds ofthis class have been isolated from natural sources, predominantly fromFusarium species (2). Depending on the concentration and thesubstitution pattern of the trichothecene, either translationalinitiation, elongation or termination are preferentially inhibited (3).

F. graminearum and F. culmorum are the two most relevant causativeagents of FHB of cereals. Whereas F. graminearum lineages present inEurope and North America produce predominantly deoxynivalenol (DON), andthe acetylated derivatives 3-acetyl-deoxynivalenol (3-ADON) and15-acetyl-deoxynivalenol (15-ADON), producers of nivalenol (NIV), whichcontains one additional hydroxyl group (FIG. 1A), dominate in Asia (4).While the toxicity of these trichothecenes is well studied in animalsystems (5), little is known about differences in phytotoxicity. Animalexposure to DON (which is also known as vomitoxin) has been shown tocause adverse health effects, the neural and immune system being themost sensitive targets (6). In contrast to high doses of DON, whichinhibit antibody production, low doses of DON act synergistically withbacterial lipopolysaccharide stimulating proinflamatory processes (6).To protect consumers, advisory levels for food have been established bythe US Food and Drug Administration, and recently action levels for DONhave been recommended by the European Community (7).

The role of DON in plant disease is still matter of discussion (8), butmost of the evidence is in favor of a function as virulence factor.Fusarium mutants with a disrupted gene involved in trichothecenebiosynthesis (trichodiene synthase, Tri5) are still pathogenic, howeverthey exhibit reduced virulence on wheat (9) due to their inability tospread from the infection site (10). DON can move ahead of the fungus ininfested plants, suggesting a role in conditioning host tissue forcolonization (11). Results of several studies indicate that the in vitroresistance of wheat cultivars towards DON correlates with FHB resistancein the field (12), which is also the case for segregating experimentalpopulations.

Increased resistance against toxic substances can be caused by severalmechanisms, ranging from reduced uptake to bypass, over-expression ormutation of the toxin target. Yet, a very prominent process seems to bemetabolic transformation often followed by compartmentation (13). Anobserved decline in the concentration of DON, which occurred in Fusariuminfected wheat in the field (14) suggested that the toxin may bemetabolized.

Acetylation of trichothecenes (e.g. DON) was suggested as a potentialresistance strategy (U.S. Pat. No. 6,346,655 B1), however, the resultingacetylated trichothecenes (e.g. Acetyl-DON=ADON) is equivalent in itsoverall toxicity to the non-acetylated form (e.g. DON), as proven byEudes et al. in a coleoptile elongation test (53). Therefore,acetylation does not result in a reduction of trichothecene toxicity.

Two wheat cultivars differing in Fusarium resistance showed differencesin their ability to form a DON metabolite, which was suspected to be aglucoside (15). Sewald et al. (16) incubated maize suspension culturewith radiolabeled DON and proved that it was primarily conjugated to3-β-D-glucopyranosyl-4-deoxynivalenol. No plant enzymes capable ofmodifying the trichothecenes or other mycotoxins by transfer of a sugarmoiety have been described so far.

Genome sequencing projects revealed the existence of a vast number ofgenes in plants that code for putative UDP-glycosyltransferases (UGT),predicted to conjugate small molecules. For instance, Arabidopsisthaliana has been shown to harbor more than 100 members of thismultigene family (17) whose functions are largely unknown.

It is therefore an object of the present invention to provide a methodfor detoxification of mycotoxins.

Suitable preparations of mycotoxins or derivatives thereof, especiallysugar-conjugate derivatives of mycotoxins are often difficult toprovide. Since stereoselectivity is crucial to this kind of products,purely chemical syntheses based on common bulk mycotoxins are oftendiffcult, if not impossible, at least on an industrial scale. Anotherobject of the present invention is therefore to provide methods forsynthesising sugar-conjugate derivatives of mycotoxins.

Therefore the present invention provides a method for detoxification ofmycotoxins wherein a mycotoxin is contacted with a glucosyltransferasein the presence of an activated glucose.

As mentioned above, there has not been any report being published in theprior art with respect to (plant) enzymes being capable of modifyingmycotoxins by transfer of a sugar molecule, and of course not withrespect to such enzymes being able to detoxify such mycotoxins, i.e.reducing the toxicity of mycotoxins to a considerable extent, e.g. bymore than 80%, preferably more than 90%, especially more than 95%, asdetermined by specific mycotoxin assays, such as inhibition of in vitroprotein synthesis.

According to the present invention it has been found thatglucosyltransferases are very specific tools for detoxification ofmycotoxins. Specificity is on the one hand given by a certainspecificity of given glucosyltransferases to only a limited number ofmycotoxins and on the other hand also specificity with respect to themode of detoxification, namely by site specific glucosylation of themycotoxins. While this specificity limits the scope of application of agiven single glucosyltransferase, the present invention provides toolsfor easily determining the specificities of any glucosyltransferase withrespect to any mycotoxin, especially trichothecene mycotoxins.Therefore, if a given enzyme is specific for a given set of mycotoxins,the present invention allows straightforward identification ofspecificities for other glucosyltransferases and mycotoxins.

Since uridine diphosphate glucose (UDP-glucose, e.g. glucose which isproduced by enzymes, such as pyrophosphorylases) is a preferredactivated biological form of glucose, a preferred embodiment of thepresent method uses UDP-glucosyltransferase as glucosyltransferase andan UDP-glucose as activated glucose (=cosubstrate), however, any formglucose, which can be utilised by a glucosyltransferase (e.g.ADP-glucose, CDP-glucose, or any other form of biologically andsynthetically activated glucose being suitable substrates for transferto mycotoxins by glucosyltransferases) can be used according to thepresent invention.

A glucosyltranferase according to the present invention is defined as a(naturally) occuring or synthetically (recombinantly) designed orproduced enzyme which is capable (as its main or as a side activity) oftransferring a glucose moiety to a substrate molecule using an activatedglucose as co-substrate thereby producing a glucosylated substratemolecule.

Preferred mycotoxins to be detoxified by the method according to thepresent invention are—due to their economic importance—trichothecenes,especially trichothecenes having a free hydroxy-group at position C₃ andtwo hydrogen groups at C₄. Especially the latter trichothecenes arespecifically glucosylated either at their position 3 or—if present—at afree hydroxy group at position 7 or at both positions. Therefore,preferred mycotoxins are deoxynivalenol, 15-acetyl-deoxynivalenol,15-acetoxyscirpendiol or mixtures thereof.

A preferred way of performing the method according to the presentinvention is the in vivo mode. This can be done using recombinant DNAtechnology and/or glucosyltransferase expression enhancing compounds.Preferred examples comprise expression of a glucosyltransferase in atransgenic plant cell (or plant tissue or whole plant) containing arecombinant glucosyltransferase and/or a recombinant regulating regionfor a glucosyltransferase, especially a mycotoxin-inducible promoter,especially of a glucosyltransferase. Preferred examples forglucosyltransferase expression enhancing compounds are substances whichup-regulate glucosyltransferases, e.g. toxins (if expression of thetransferase is controlled by toxin-inducible promoters). In principle,any cell which can be genetically manipulated so that it expresses arecombinant (i.e. a gene which has been artificially introduced intothis cell or its precursor and which naturally is not present at thissite of the genome of the cell) plant glucosyltransferase gene can beused for this purpose. Preferred embodiments are transgenic bacteria,yeasts, baculovirus infected insect cells, etc. expressing a plantglucosyltransferase gene.

In an industrial process for detoxification of mycotoxins, theglucosyltransferase can advantageously be immobilised (according tostandard immobilisation techniques) to a solid surface and a mycotoxincontaining solution or suspension can then be contacted with thisimmobilised glucosyltransferase.

Preferred examples of glucosyltransferases to be used according to thepresent invention are UDP-glucosyltransferases corresponding tosubfamily 73C of Arabidopsis thaliana, especiallyUDP-glucosyltransferases 73C4 and 73C5.

According to another aspect, the present invention enables a method forproducing glucosylated mycotoxins wherein a mycotoxin is contacted witha glucosyltransferase in the presence of an activated glucose.Therewith, the above mentioned enzyme specificities can be properly usedfor producing specifically glucosylated mycotoxin derivatives. If amycotoxin is glucosylated in a non-enzymatic (“chemical”) way, a crudemixture of glucosylation products is obtained (each free hydroxy groupis in principle an acceptor site for glucosyl residues). Acetylation asprotecting means for such groups has also its drawbacks with respect tospecificity (also all hydroxy groups are acetylated). In using substrateand site specificity, the present invention provides a method forproducing well defined glucosylated derivatives. If more than onehydroxy group is glucosylated or if more than one position is capable ofbeing glucosylated by a given glucosyltransferase, such a limited numberof products (e.g. 2 to 5) may easily be separated by suitable separationmeans such as HPLC (for example, DON, being glucosylated at positionnumber 3, is easily separatable from DON, being glucosylated at position7).

The preferred method steps described above are also applicable for thisaspect of the invention.

According to another aspect, the present invention relates to transgenicplants or a transgenic (plant) cells containing a recombinantglucosyltransferase and/or a recombinant expression regulating element,especially a promoter region for a glucosyltransferase. These plants or(plant) cells may be used in a method according to the presentinvention.

The present invention also relates to a genetically modified cell ororganism, especially a plant or plant cell, comprising a non-naturallyoccurring (transgenic) glucosyltransferase in its genome and beingmycotoxin resistant, especially trichothecene resistant, due to thepresence of such a glucosyltransferase as transgene. Alternatively, anendogeneous glucosyltransferase in a cell may be subjected to adifferent, transgenic expression regulating region or element, such as apromoter, leading to an enhanced expression of the endogeneousglucosyltransferase (i.e. an enhanced expression activity of ahomologous glucosyltransferase due to transgenic promoters). Also suchcells show enhanced resistance against mycotoxins as described herein.Such cells and plants can be produced by applying standard methods tothe teachings of the present invention (see e.g. FIG. 6B and theexamples as well as e.g. (54)).

Preferred cells according to the present invention are also yeast cells.Since yeast cells may have a limited uptake capability, preferred yeastcells according to the present invention are designed or selected for anincreased mycotoxin-uptake capability compared to the wild type orstarting strain. Specifically preferred according to the presentinvention are yeasts in which one or more, preferably three or more, ABCtransporters are reduced or inactivated in their activity, especiallypdr5. Such ABC transporter mutants are characterized by an enhanceduptake of mycotoxins. Preferably, the cells according to the presentinvention have a deletion in pdr5, pdr10, pdr15, snq2, yor1, ayt1 orcombinations of these deletions.

These cells may preferably be used for detoxification of mycotoxincontaining solutions or suspensions, especially for detoxification oftrichothecenes in agriculture and beer production; and in the productionof glucosylated mycotoxins, especially glucosylated DON or 15-ADON.

The present invention is further illustrated by the following examplesand the figures, yet without being restricted thereto.

FIGURES

FIG. 1. Spectrum of trichothecene resistance of yeast expressingArabidopsis thaliana UDP-glucosyltransferases of subfamily UGT73C. A,Structure and numbering of the ring system of class B trichothecenesused for resistance testing. The different residues at variablepositions R1-R4 are indicated in the table (OAc: —OCOCH₃, OBe:—OCOCH═CHCH₃ (Z)). The relevant R1 is underlined (OH in DON, alreadyblocked by acetylation in 3-ADON). Increased resistance conferred byDOGT1 expression is indicated by plus, lack of protection by minus. B,Genomic organisation of the UGT gene cluster on chromosome II (subfamily73C) containing DOGT1 (UGT73C5, locus At2g36800). The DON detoxifyingDOGT1 and 73C4 are shaded in gray. C, Yeast strains were spotted on YPDplates containing the indicated amount of toxin (ppm: mg/l). The strainsare wild-type YZGA515 without plasmid (wt), this strain transformed withempty vector (c), and the transformants expressing the myc-tagged 73CUGTs (C5=DOGT1, underlined, spotted in duplicate). Resistance isconferred by plasmids leading to overexpression of C5 and C4. D, Westernblot analysis of the yeast strains used for resistance testing. TheN-terminal c-Myc epitope-tag introduced into the respective 73C UGTfamily members is detected (C5=DOGT1, underlined). A transformantcontaining the empty expression vector was used as control.

FIG. 2. Amino acid alignment of plant UDP-glucosyltransferases with highamino acid similarity to DOGT1. The regions implicated (Ref. 38) inacceptor substrate binding (dottet) and the UGT consensus sequence motif(dashed) are indicated by boxes below the sequences. The triangle abovethe sequence in the hypothetical acceptor binding region marks thelysine 136 in DOGT1 which has been altered by in vitro mutagenesis. Thegenbank accession numbers of the predicted proteins are: ADGT-9,glucosyltransferase-9 of Vigna angularis (AB070752); TOGT1,Phenylpropanoid:glucosyltransferase 1 of Nicotiana tabacum (AF346431);IS5a of Nicotiana tabacum (U32644); putative glucosyltransferase ofOryza sativa (AP002523); Twil of Lycopersicon esculentum (X85138);Betanidin-5-O-glucosyltransferase of Dorothenathus bellidiformis(Y18871).

FIG. 3. DOGT1 expression is developmentally regulated and induced by DONand stress response related compounds. A, GUS-staining of seedlingshomozygous for a transcriptional DOGT1 promoter-GUS fusion. Upper row:three days after germination (3 DAG) the expression of the fusionprotein is restricted to the vasculature of root and hypocotyl and themeristematic region of the root tip. Middle: Later in development (10DAG) staining in the root vasculature diminishes except for regionswhere lateral roots are formed. Lower row: In aerial parts of adultplants DOGT1 expression is restricted to petals of flowers andabscission zones. B, DON treatment (5 ppm for 4 h) of seedlings (14 DAG)expressing the translational GUS-fusion induces expression of the GUSreporter. Both samples were stained for 2 hours. C, Semiquantitativereverse transcriptase PCR analysis of induction of expression of DOGT1,UGT73C4 and UGT73C6 following treatment with DON (5 ppm), SA (100 μM),JA (50 μM) and ACC (2 μM). UBQ5 was used as an internal control.

FIG. 4. The N-terminal part of DOGT1 is essential for its ability todetoxify DON. A, Amino acid alignment of DOGT1 and its closest homologueUGT73C6, which is not protecting against DON (black: amino acidsidentity). The conserved EcoRI restriction site was used to generatechimaeric UGTs consisting of the N-terminus of one and the C-terminus ofthe respective other gene. B, Yeast transformants were spotted induplicate on YPD medium containing the indicated concentrations of DON.Upper left: transformants expressing the chimaeric enzyme consisting ofthe UGT73C6 N-terminus and DOGT1 C-terminus (short: C6-DOG). Upperright: Strains expressing the chimaeric protein consisting of DOGT1N-terminus and UGT73C6 C-terminus (DOG-C6). Lower row: transformantsexpressing resistance conferring DOGT1 (left), and inactive 73C6(right). The hybrid containing the N-terminal part of DOGT1 is resistantto higher DON concentrations than the strain expressing the other hybrid(C6-DOG) or UGT73C6.

FIG. 5. Fragmentation pattern of synthesized DON-glucosides and aDON-metabolite formed in yeast cells expressing DOGT1 in massspectrometry. A, The synthesized DON-3-O-glucoside yields a fragment of427.2 m/z in the linear ion trap (MS/MS/MS). B, this fragment was notproduced by the synthesized DON-15-O-glucoside sample as a separation ofthe —CH₂OH group at C₆ is prevented by the glucose moiety present at thehydroxyl group. C, The DON-metabolite formed in yeast expressing DOGT1eluted in the HPLC with the same retention time as the DON-3-O-glucosideand showed the fragmentation pattern corresponding to theDON-3-O-glucosid reference substance.

FIG. 6. glucosylation of DON reduces toxicity in vitro and in vivo. A,Structure of the proposed reaction product: DOGT1 catalyses the transferof glucose from UDP-glucose to the 3-OH position of DON. B, Comparisonof the inhibition of in vitro protein synthesis of wheat ribosomes byDON and DON-3-O-glucoside, determined using a wheat germ extract basedcoupled transcription/translation system. Luminescence was measured andexpressed as % luciferase activity of control samples without toxin. C,Western blot analysis of A. thaliana lines homozygous for anoverexpression construct encoding c-Myc-tagged DOGT1. A schematicdrawing of the plant transformation vector is shown below (GENT:gentamycin resistance used as selectable marker; 2×35S duplicatedpromoter of cauliflower 33S transcript (see Experimental Procedures fordetails)). Col-0 was used as a negative control. 2 lines with high and 2lines with low levels of c-Myc-DOG1 protein are shown. D, Seedgermination on DON containing MS medium. Compared to wild-type (Col-0)transgenic Arabidopsis lines expressing high amounts of DOGT1 (e.g. line1319/2) exhibit enhanced resistance.

EXAMPLES

For illustrating the present invention, in the examples hereinafter thecloning of an Arabidopsis UGT by functional expression in yeast and itscharacterization is described. This enzyme is able to detoxify theFusarium mycotoxin DON and its acetylated derivative 15-ADON. Themethods used in these examples are also directly applicable to otherglucosyltransferases and mycotoxins without undue experimental burden.

EXPERIMENTAL PROCEDURES

Yeast strains. The yeast strains used in this work are derived fromYPH499 (Mat a, ade2-101oc, his3-Δ200, leu2-Δ1, lys2-801a, trp1-Δ1,ura3-52) (18). The relevant genotype of YZGA452 is pdr5Δ::TRP1,pdr10Δ::hisG, snq2Δ::hisG, yor1Δ::hisG. YZGA515 (pdr5Δ::TRP1,pdr10Δ::hisG, pdr15Δ::loxP-KanMX-loxP, ayt1Δ::URA3) was constructed bydisruption of the acetyltransferase AYT1 in strain YHW10515.

Plant material and growth conditions. A. thaliana experiments wereconducted with the wild-type ecotype Columbia-0 (Col-0). For propagationseeds were sterilized, plated on standard MS growth medium (19)supplemented with 1.0 % sucrose and 1.0% phytagar (Life Technologies)and subjected to a 2 day dark treatment at 4° C. to synchronizegermination. The seedlings were grown for 2 weeks in a controlledenvironment of 16 h/8 h light-dark cycle (140 μmol m⁻² sec⁻¹ whitelight) at 22° C. before they were transferred to soil and grown at 20°C. and 55% humidity under continuous white light.

Arabidopsis thaliana cDNA library screen in yeast. The ATP-bindingcassette (ABC) transporter deficient Saccharomyces cerevisiae strainYZGA452, which is hypersensitive to DON, was transformed with an A.thaliana cDNA library constitutively expressed under the control of thephoshoglucerate kinase (PGK1) promoter (20). A total of 10⁷transformants were selected on minimal medium lacking uracil andtransferred to media containing 180 ppm DON, sufficient to completelyinhibit growth of yeast transformed with the empty library plasmid.Colonies that showed resistance were isolated and from candidates thatformed single colonies on toxin containing media, the plasmid dependencyof the phenotype was tested by plasmid DNA preparation andretransformation of YZGA452. The NotI fragment containing the cDNAinsert of the candidate (which was named DON glucosyltransferase 1,DOGT1) was subcloned into pBluescript SKII+ (Stratagene) and sequenced.

Constitutive expression and immunodetection of theDON-glucosyltransferase (DOGT1) and close homologues in yeast. Theintronless open reading frames (ORFs) of DOGT1 (UGT73C5, locusAt2g36800) and 5 of its closest homologues (UGT73C1, At2g36750; UGT73C2,At2g36760; UGT73C3, At2g36780; UGT73C4, At2g36770; UGT73C6, At2g36790)were PCR amplified (Triple Master PCR System, Eppendorf) from genomicDNA using gene specific primers containing flanking HindIII and NotIrestriction sites at the 5′ and 3′ ends, respectively

-   (DOGT1, fw: 5′-ACTAAGCTTGGAATCATGGTTTCCGAAACA-3′, rv:    5′-AAGCGGCCGCATACTCAATTATTGG-3′; 73C1, fw:    5′-CTAAGCTTGGAATCATGGCATCGGAATTTCG-3′, rv:    5′-TAGCGGCCGCATTCATTTCTTGGGTTGTTC-3′;-   73C2, fw: 5′-CTAAGCTTGGAATCATGGCTTTCGAGAAGACC-3′, rv:    5′-TAGCGGCCGCATTCAACTCTTGGATTCTAC-3′;-   73C3, fw: 5′-CTAAGCTTGGAATCATGGCTACGGAAAAAACC-3′, rv:    5′-TAGCGGCCGCATTCATTCTTGAATTGTGC-3′;-   73C4, fw: 5′-CTAAGCTTGGAATCATGGCTTCCGAAAAATC-3′, rv:    5′-TAGCGGCCGCATTCAGTTCTTGGATTTCA-3′;-   73C6: 5′-CTAAGCTTGGAACATGTGTTCTCATGATCCT-3′, rv:    5′-TAGCGGCCGCATTCAATTATTGGACTGTGC-3′). The PCR products were cloned    into the HindIII+NotI cloning sites of the yeast expression vector    pYAK7 (PADH1-c-Myc-PDR5 LEU2 2μ), replacing the PDR5 gene. The    vector pYAK7 was constructed by first inserting the double stranded    linker    5′-GGATGCCCGAACAAAAGTTAATTTCAGAAGAGGACTTATCAAAGCTTGAGGCCTCGCGA into    the SmaI site of vector pAD4Δ (21), thereby generating the    N-terminal c-Myc epitope and a HindIII site, into which a genomic    HindIII fragment containing the yeast PDR5 was inserted in frame.

The tagged UGT constructs were verified by sequencing and used totransform the yeast strain YZGA515. The empty vector (HindIII+NotIdigested and religated pYAK7) was used as a control. Transformants wereselected on minimal media lacking leucine. Exponentially growingcultures were diluted to OD600 0.05 and spotted on YPD media containingincreasing concentrations of different trichothecenes. The toxins usedwere: DON, 3-ADON, 15-ADON, NIV, trichothecin (TTC), T-2 toxin, HT-2toxin, diacetoxyscirpenol (DAS) and veruccarin A (VA). With theexception of DON, 3-ADON and NIV, which were obtained from BiopureReferenzsubstanzen GmbH (Tulln, Austria), mycotoxins were purchased fromSigma and stored at −20° C. dissolved in 70% ethanol.

For immunodetection, the extraction of proteins from yeast cells wasperformed as described by Egner et al. (22). Western blot analysis wasconducted with a primary mouse anti c-Myc antibody (1:5000, clone 9E10,Invitrogen).

Domain shuffling. The ORFs including the N-terminal c-Myc tag of DOGT1and its closest homologue UGT73C6 were isolated from the yeastexpression vectors by SmaI+NotI digestion, cloned into vectorpBluescript SKII+ (which was cut with XhoI and treated with Klenowenzyme, and subsequently digested with NotI). The resulting plasmidswere digested with HindIII and a conserved EcoRI site present in bothgenes that cleaves DOGT1 at nucleotide position 565 (73C6 at 568).Hybrids were constructed by ligation of the N-terminal part of one geneto the C-terminal part of the other. The resulting genes were moved backinto the yeast expression vector pYAK7 using the HindIII and NotIrestriction sites. Yeast strain YZGA515 was used as a host to test theconstructs for altered detoxification abilities.

Site directed mutagenesis. Mutations were constructed by overlapextension PCR (23) using overlapping mutant primers DOG-K136E-fw(5′-TACAAGCGAAATCGCCAAGAAGTTCA-3′) and DOG-K136E-rv(5′-CTTCTTGGCGATTTCGCTTGTATAAG-3′) and flanking primers DOGIpYAK7-fw-a(5′-ACTAAGCTTGGAATCATGGTTTCCGAAACA-3′) and DOG-EcoRI-rv(5′-TCTTGTGAATTCAACTCTATC AGGA-3′) for mutagenesis of DOGT1, and mutantprimers 73C6-E137K-fw (5′-TACAAGCAAAATCGCC AAGAAGTTCAA-3′) and73C6-E137K-rv (5′-ACTTCTTGGCGATTTTGCTTGTATT-3′) and flanking primers73C6pYAK7-fw (5′-TAAGCTTGGAATCATGTGTTCTCATGATCCT-3′) and DOG-EcoRI-rvfor 73C6 mutagenesis. The resulting PCR products were cloned asHindIII+EcoRI fragments into the corresponding genes present in vectorpBluescript SKII+. After sequencing, the ORFs were moved as HindIII+NotIfragments back into the yeast expression vector pYAK7 (replacing thePDR5 gene) and the resulting plasmids were transformed into strainYZGA515 to analyze the detoxification properties of the engineered UGTs.

Synthesis of 3-β-D-Glucopyranosyl-4-deoxynivalenol and15-β-D-Glucopyranosyl-4-deoxynivalenol. To obtain reference material forHPLC and TLC, DON-3-glucoside and DON-15-glucoside were synthesized in2-step reactions. In the first step, 15-Acetyl-DON or 3-Acetyl-DON weremodified with 1ββ-Bromo-1-deoxy-2,3,4,6-tetra-O-acetyl-α-D-glucopyranose(acetobromoglucose) in toluene with CdCO₃ as catalyst to yield theDON-glucoside-acetates (24). Gentle hydrolysis of the acetates to theglucosides was performed in the second step using a strong basic anionexchanger (DOWEX1×2-400, Aldrich, USA). After checking the progress ofthe reaction with TLC (mobile phase toluene/ethyl acetate 1:1, v/v), theDON-glucosides were cleaned up using flash chromatography over silicagel with 1-butanol/1-propanol/ethanol/water (2:3:3:1, v/v/v/v). Furtherpurification of the substances was performed with HPLC, by means of aRP-18 Aquasil column (Keystone, Waltham, USA) using acetonitrile/water(10:90, v/v) at 22° C.

The synthesized DON-derivatives were characterized in the negativeelectrospray interface (ESI) mode. LC-MS/MS analysis was performed on aQTrap-LC-MS/MS system (Applied Biosystems, Foster City, USA) equippedwith ESI and a 1100 Series HPLC system (Agilent, Waldbronn, Germany).Chromatographic separation was achieved on a 150 mm×4,6 mm i.d., 3 μm,Aquasil RP-18 column (Keystone, Waltham, USA) at 22° C. usingmethanol/water (28:72, v/v). The flow rate was set to 0.3 ml/min. TheESI interface was used in the negative ion mode at 400° C. with CUR 20psi, GS1 30 psi, GS2 75 psi, IS −4200 V, DP −46 V, EP −9 V, CE −30 eV,CAD high, LIT fill time 50 ms, Q3 entry barrier 8 V.

Isolation and analysis of DON metabolites in vivo. To elucidate thechemical structure of DON metabolites that result from enzymatictransformation of the mycotoxin by DOGT1, a highly tolerant strain wasconstructed and the DON-metabolite was extracted from toxin treatedcells for subsequent HPLC analysis.

The yeast strain YZGA515 was transformed with c-Myc-tagged DOGT1 underthe control of the constitutive ADH1 promoter and in addition withplasmid pRM561. This plasmid contains a mutant version of the ribosomalprotein L3 (RPL3) of Saccharomyces cerevisiae that substantiallyincreases DON resistance when expressed in yeast. Transformants wereselected on minimal media lacking leucine and adenine.

The obtained yeast strain was grown to an OD₆₀₀ 0.7 in selective medium(SC-LEU-ADE). Cells were transferred to YPD medium (10% glucose)supplied with additional adenine, grown for 2 h at 30° C., harvested bycentrifugation and diluted to an OD₆₀₀ of 3.0 in YPD. DON was added to 5ml of culture starting with 200 ppm DON. After 3 h the concentration wasincreased to 400 ppm, after 6 h to 600 ppm, and after 9 h to 1.000 ppm.The cells were incubated for further 15 h at 30° C., before they wereharvested, washed three times with ice-cold water, extracted in 2.5 mlof methanol/water (4:1) and sonicated. After centrifugation thesupernatant was filtered through a glass microfibre filter (Whatman 1822025). 500 μl of the yeast extracts were concentrated to dryness under aconstant stream of nitrogen and dissolved in 100 μl HPLC-grade water.

Heterologous expression of DOGT1 in Escherichia coli. The DOGT1 proteinwas expressed in Escherichia coli XL1-blue as a GST fusion. The DOGT1gene was released from the yeast expression vector by HindIII digestionand Klenow fill in, followed by a NotI digest. The resulting fragmentwas cloned into the SmaI+NotI sites of the GST gene fusion vectorpGEX-4T-3 (Amersham Pharmacia). Recombinant fusion protein was purifiedusing glutathione-coupled Sepharose (Amersham Pharmacia) according tothe manufacturer's instructions.

To test the effect of the N-terminal GST tag on activity, the geneencoding the fusion protein was PCR amplified using DNA polymerase withproof reading activity (Pfu polymerase, MBI) and the fusion proteinspecific primers GSTDOGpYAK7-fw (5′-TCACCCGGGAAACAGTAATCATGTCC-3′) andGSTDOGpYAK7-rv (5′-CGAGGCAGATCGTCAGTCAGTC-3′). The PCR product wascloned HindIII+NotI into the yeast expression vector pYAK7.DON-detoxification ability was tested by expression in YZGA515 andapplication to toxin containing media as described earlier.

Enzyme assays. The glucosyltransferase activity assay mix contained 1 μgof recombinant GST-fusion protein, 10 mM 2-mercaptoethanol, 50 mMTris/HCl pH 7.0, 0.5 mM radioactive labeled UDP-[¹⁴C] glucose (4.4 *10³cpm, NEN Life Science Products, USA), 0.01% BSA and 1 mM of acceptorsubstrate (dissolved in DMSO in 20 mM stock solutions). The reactionswere carried out in volumes of 20 μl at 30° C. for 1 h, stopped byadding 2 μl trichloroacetic acid (240 mg/ml), frozen and stored at −20°C.

Analysis of reaction products was performed by TLC. An aliquot of eachsample was spotted on a silica-gel plate (Kieselgel 60; Merck) anddeveloped with a mixture of 1-butanol/1-propanol/ethanol/water (2:3:3:1,v/v/v/v). The intensity of each radioactive spot was determined using aphosphoimager (STORM 860 system, Molecular Dynamics). The plates wereadditionally stained with panisaldehyde (0.5% in methanol /H₂SO₄/aceticacid, 85:5:10, v/v/v).

Inhibition of wheat ribosomes in vitro. To analyze whether the3-β-D-Glucopyranosyl-4-deoxynivalenol is less phytotoxic than itsaglucon, a wheat germ extract coupled in vitro transcription/translationsystem (TNT Coupled Wheat Germ Extract, T3, Promega) was used. Afterperforming transcription/translation reactions for 25 minutes accordingto the manufacturers instructions in the presence of either DON,purified DON glucoside (1 μM, 2.5 μM, 5 μM, 10 μM and 20 μM) or water asa control, the activity of the firefly luciferase reporter wasdetermined (Luciferase Assay System, Promega) using a luminometer(Victor 2, Wallac).

Plant treatment with different stress response related compounds forexpression analysis. For Reverse Transcription (RT)-PCR analysis of mRNAexpression of DOGT1 following treatments with DON, salicylic acid (SA),jasmonic acid (JA) and 1-aminocyclopropyl-carbonic acid (ACC) seedlingswere grown for 2 weeks on vertical MS plates (0.8% phytagar) before theywere transferred to liquid MS media. The plants were incubated for 48 hon an orbital shaker (50 rpm) before adding 5 ppm DON, 200 μM SA, 2 μMACC or 50 μM JA. The compounds were kept in stock solutions dissolvedeither in 70% ethanol or in DMSO. Treatments with ethanol and DMSO wereperformed as controls. Plants were harvested at different time points,ground in liquid nitrogen and stored at −70° C. until RNA extraction wasperformed.

Analysis of mRNA expression of DOGT1 and close homologous by ReverseTranscriptase-PCR. Total RNA was isolated from plant tissue ground inliquid nitrogen with Trizol Reagent as recommended by the manufacturers(Gibco BRL Life Technologies). RNA was quantified photometrically andvisually on a denaturing RNA gel analyzing 5 μg of total RNA.

cDNA was synthesized from 1 μg of total RNA (digested with DNa-seI) with500 ng of a 18-mer oligo(dT) and the reverse transcriptase SuperScript(Gibco BRL Life Technologies). PCR was performed with approximately 2 μlof the 1:20 diluted cDNA using primers that amplify 200 to 400 bp largefragments located in the C-terminal part of the genes to be analyzed(DOGRT-fw: 5′-ATCCGGGGTTGAACAGCCT-3′, DOGRT-rv:5′-TCAATTATTGGGTTCTGCC-3′; 73C4RT-fw: 5′-GGAGAAAATAGGAGTGTTA-3′,73C4RT-rv: 5′-TCAGTTCTTGGATTTCACT-3′; 73C6RT-fw:5′-GAGAAACTGGTCGTACAA-3′, 73C6RT-rv: 5′-TCAATTATTGGACTGTGCT-3′; UBQ5-U:5′-GTCCTTCTTTCTGGTAAACGT-3′, UBQ5-D: 5′-AACC CTTGAGGTTGAATCATC-3′). Tocompare relative amounts of transcripts in the samples, DNA fragments ofthe UBQ5 were first amplified and normalized sample volumes based on theamount of products corresponding to the UBQ5 transcripts were used forPCR.

Cloning of plant overexpression constructs and GUS-fusion constructs.For constitutive overexpression of c-Myc-tagged DOGT1 protein inArabidopsis, the vector pBP1319 was constructed. It is derived from amodified version of the plant expression vector pPZP221 (25). Thepromoter cassette consisting of two copies of the 35S-promoter and thepolyadenylation signal of CaMV strain Cabb B-D was used from the vectorp2RT, a modified version of pRT100 (26).

The c-Myc-tagged DOGT1-fragment was released by SmaI+NotI digestion(Klenow filled) from the yeast expression vector and cloned intopBluescript SKII+, which was cut with ClaI+SmaI and treated with Klenowenzyme. In the next step, the gene was excised from the resultingplasmid as SalI+BamHI fragment and inserted in the XhoI+BamHI sites ofp2RT. The obtained 2×35S c-Myc-DOGT1 cassette was isolated by PstIdigestion and cloned into the unique PstI site of pPZP221 after themultiple cloning site in that vector had been destroyed by digesting theplasmid with EcoRI+SalI, filling the sites with Klenow and religatingit. In the resulting vector pBP1319, the 2×35S c-Myc-DOGT1 cassette isorientated in the opposite direction than the 2×35S gentamycinresistance marker.

For construction of a transcriptional DOGT1-GUS fusion, the GUS vectorpPZP-GUS.1 which originates from pPZP200 and contains the GUS gene frompBI101.1 (inserted HindIII+EcoRI into the MCS), was used (27). The DOGT1promoter region was PCR amplified from genomic DNA using DNA polymerasewith proof reading activity (Pfu Polymerase, MBI) and specific primers(DOGP-GUS-fw: 5′-GTTAAAAGCTTACATGTGCATTACGGTCTGTGTGAATA, DOGP-GUS-rv:5′-TTTCGGATCCCATG ATTCAACCTTAGTAAGAAACTCTC). The resulting product wascloned in frame with the GUS gene HindIII+BamHI into the pPZP-GUS.1vector. Constructs were confirmed by DNA sequencing.

Generation and analysis of transgenic A. thaliana. For all planttransformations, the recA deficient Agrobacterium tumefaciens strainUIA143 (28) which harbors the helper plasmid pMP90 (29) was used. A.thaliana was transformed applying the floral dip technique (30). Theprogeny of 15 independent transformants was selected through threegenerations to obtain homozygous lines.

For immunodetection of c-Myc-tagged DOGT1, about 200 mg-500 mg of plantmaterial were homogenized in liquid nitrogen. 300 μl of extractionbuffer (200 MM Tris-HCl pH 8.9; 200 mM KCl; 35 mM MgCl₂; 12.5 mM EGTA;15 mM DTT; 0.6 mM sorbitol) and 15 μl of proteases inhibitor cocktail(Sigma, #9599) were added to the still frozen samples and the mixturewas incubated under vigorous shaking for 15 minutes at 4° C. Aftercentrifugation (14.000 rpm for 15 minutes at 4° C.), 200 μl of thesupernatants were transferred into fresh tubes and stored at −20° C.Equivalent amounts of protein (50 μg) were used for Western blotanalysis which was carried out using primary anti c-Myc antibodypurified from hybridoma supernatant (clone 9E10).

For analysis of DON resistance, seeds of homozygous lines exhibitinghigh DOGT1-expression and Col-0 as control were germinated on MS mediacontaining different concentrations of DON (5-30 ppm). Seedlings weregrown for 5 weeks before the phenotype was documented. GUS activity wasanalyzed by staining seedlings or organs of adult plants in X-Glucsolution for 2 to 4 h at 37° C. (31).

RESULTS

Isolation of the DON-glucosyltransferase (DOGT1) by heterologousexpression in yeast. An unbiased functional screen based on heterologousexpression of cDNAs in yeast was set up with the goal to identify plantgenes that contribute to resistance against mycotoxins. Wild-typeSaccharomyces cerevisiae is highly resistant to deoxynivalenol (DON). Inorder to reduce the amount of toxin necessary for the screen, a straindeficient in four ABC transporters was generated, which are to a largeextent responsible for pleiotropic drug resistance in yeast (32). StrainYZGA452 (snq2Δ::hisG pdr5Δ::TRP1 pdr10Δ::hisG yor1Δ::hisG) ishypersensitive to a wide range of different xenobiotic substances andnatural products, including DON. YZGA452 was transformed with a cDNAexpression library of A. thaliana (20), where cDNAs are constitutivelyexpressed under the control of the yeast phosphoglucerate kinasepromoter. Ten million transformants were generated and diluted pools oftransformants were plated on DON containing medium. After selection ofDON resistant yeast colonies and confirmation of plasmid dependency ofthe phenotype, the insert was subcloned and sequenced.

DOGT1 is a member of the UDP-glucosyltransferase family of A. thalianaand exhibits high similarity to salicylic acid and wound inducible genesof other species. The cDNA conferring resistance had a size of 1.75 kband contained an open reading frame of 1488 bp length encoding aputative uridine diphosphate (UDP)-glucosyltransferase. The identifiedDON-glucosyltransferase (DOGT1) corresponds to gene UGT73C5 and belongsto subfamily 73C, part of group D of UDP-glucosyltransferases (UGTs) ofA. thaliana (33). Arabidopsis UGTs constitute a very large gene family,that has been divided into 14 distinct groups, believed to haveoriginated from common ancestors (17). DOGT1 is located in a cluster(FIG. 1B) together with five other members of subfamily 73C onchromosome II (BAC clone F13K3, At2g36800). All six tandemly repeatedgenes contain no introns, and are highly similar to each other (77-89%identity at the amino acid level). The similarity is also very high inthe intergenic promoter regions.

A database search with the DOGT1 amino acid sequence revealed highsimilarity to glucosyltransferases from tobacco (TOGT1, ref. 34; Is5aand Is10a, ref. 35) and tomato (Twi-1, ref. 36), the expression of whichhas been shown to be elevated following treatment with salicylic acid(SA), fungal elicitors or wounding (34, 36, 37), and to the betanidin5-O-glucosyltransferase of Dorotheanthus bellidiformis (38). Twoputative, uncharacterized glucosyltransferases from Vigna angularis(ADGT-9) and Oryza sativa with homology to DOGT1 were also included inthe amino acid alignment shown in FIG. 2. Regions of high similaritywere observed in both amino- and carboxy-terminal domains of the deducedamino acid sequences. Indicated in FIG. 2 are the hypothetical acceptorsubstrate binding region (38) and the UGT consensus sequence (33).

The expression of DOGT1 is developmentally regulated and induced by DONand other stress response related compounds. To investigate whether theexpression of DOGT1 is regulated in a similar fashion as described forthe related genes of other plant species, the ORF of the β-glucuronidasereporter gene was placed behind the DOGT1 promoter (P_(DOGT1)-GUS). Thetissue specific expression of the transcriptional GUS fusion wasexamined histochemically in transgenic Arabidopsis homozygous for thefusion gene. The results shown in FIG. 3 demonstrate that DOGT1expression is regulated developmentally and is overall rather low. Inseedlings, GUS activity was observed to be root and hypocotyl specific,with the strongest expression in the vascular system, in meristematictissue of the root tips (in the primary root as well as in lateralroots) and in the vasculature of the hypocotyl right after germination.Staining in the vasculature decreased significantly later in developmentand a patchy staining pattern appeared in epidermal root cells. In adultplants GUS activity was detected in late stages of flower development inpetals and in abscission zones (FIG. 3A).

Exposure of seedlings to either DON (5 ppm for 4 h) or the ethyleneprecursor aminocyclopropylcarbonic acid (ACC, 2 μM for 1 h) was found toinduce P_(DGT1)-GUS expression (FIG. 3). No induction of expression ofthe reporter was detected upon SA-treatment (200 μM for 12 h) ortreatment with jasmonic acid (JA, 50 μM for 1 h). Semiquantitativereverse transcriptase PCR was applied to validate the results obtainedfrom GUS reporter analyses by detecting changes in mRNA levels of DOGT1,73C4 and 73C6 following treatment with the same concentrations of DON,SA, ACC and JA as used before.

As shown in FIG. 3C the results of the reverse transcriptase PCR confirmthe DON inducible expression of DOGT1 previously observed with thereporter construct, and furthermore show that also the other two testedUGTs are DON inducible. An increase in the amounts of transcripts wasobserved already after 1 h of incubation with the toxin, reaching a peakafter 4 h and declining again between 6 and 12 h. Interestinglu, 73C6showed a stronger induction of expression by DON than DOGT1 or 73C4although the data shown below indicate that it does not accept the toxinas a substrate. After SA treatment, DOGT1, 73C4 and 73C6 expression wasevident at low levels at 4 h and slightly stronger at 12 h. It has to benoted that the applied concentration of 200 μM SA induced the expressionof the 3 genes rather weakly. Jasmonic acid as well as treatment withACC also lead to weak induction of expression of DOGT1 and UGT73CGapparent already after 1 h of treatment, but rapidly declining with notranscript accumulation detectable anymore after 2 h of exposure to thecompounds (FIG. 3C).

Phenotypic determination of the spectrum of trichothecene resistance inyeast. Trichothecene toxicity in animals depends on the hydroxylationpattern, as well as on the position, number and complexity ofesterifications (5). The basic trichothecene structure and numberingsystem of carbon atoms is shown in FIG. 1A. Members of the subclass B(e.g. DON and NIV), contain a keto group at carbon C-8, while type Atrichothecenes, (e.g. the highly toxic T-2 toxin produced by F.sporotrichoides) do not. Extremely toxic are also macrocyclictrichothecenes, like veruccarin A, which contain a macrocyclic ring withester bonds bridging carbon 4 and carbon 15. Yeast pdr5 mutants arehypersensitive to all the trichothecenes tested so far, allowing us toinvestigate the ability of DOGT1 and other genes in the cluster toconfer resistance to various members of the trichothecenes.

The high similarity of UGT73C1, C2, C3, C4, C5 (DOGT1) and C6 and theirclustered appearance on the chromosome suggests that they have evolvedvia gene duplication from one ancestral gene and might therefore haverelated enzymatic properties. To analyze possible similarities in theirfunction, the six ORFs were amplified with specific primers andexpressed in yeast as fusion proteins with an N-terminal c-Myc epitopetag. Comparison of transformants expressing tagged or untagged DOGT1showed that the epitope did not interfere with the DON-protectiveactivity. The yeast transformants representing the full gene set of thecluster were spotted on media containing increasing concentrations ofvarious trichothecenes. Transformants containing the empty expressionvector were used as controls.

The results shown in FIG. 1 revealed that UGT73C4, like DOGT1, confersDON resistance. Although C4 has only the second highest similarity (79%identity to DOGT1), it had the ability to detoxify DON and 15-ADON (FIG.1C), but failed to confer resistance to 3-ADON, NIV, TTC, HT-2-toxin,T2-toxin, DAS and VA. In contrast, UGT73C6 which exhibits with 89%identity on amino acid level the highest similarity did not protectagainst any of the tested trichothecenes. Likewise, 73C3 was not able toconfer resistance to the tested mycotoxins although all of the fourproteins were expressed to a similar extent in yeast. The 73C1 and 73C2constructs did also not enhance toxin resistance, however this couldalso be due to lower recombinant protein levels present in the yeasttransformants (FIG. 1D).

DON detoxification specificity is located in the N-terminal part ofDOGT1. The different characteristics of the closely related enzymesdespite their high sequence similarity opened the possibility toelucidate features of the proteins which are crucial for DONrecognition. An EcoRI site present in both DOGT1 and 73C6 (see FIG. 4A)was used to construct hybrids containing the N-terminal part of one andthe C-terminal part of the respective other gene. Expression in yeastand exposure to DON containing media revealed that the N-terminus ofDOGT1 (consisting of the first 189 aa) is essential for the ability todetoxify DON (FIG. 4B).

A lysine residue at position 136 in DOGT1 is conserved in the detoxifier73C4 but replaced by other amino acids in the two nondetoxifyinghomologues C3 and C6. Site directed mutagenesis was used to introducemutations into DOGT1 and the UGT73C6 leading to a change of the lysineresidue 136 in DOGT1 to glutamic acid, which is present at thecorresponding position in C6 (amino acid 137). The mutant DOGT^(K136E)protein protected with the same efficiency as DOGT1, demonstrating thatK136 is not essential for the DON detoxification ability. Also thereverse construct 73C6^(E137K) remained inactive.

In vivo and in vitro analysis proof that DOGT1 catalyses the transfer ofglucose from UDP-glucose specifically to the 3-OH position of DON. Theprotection against 15-ADON and the inability to confer resistanceagainst 3-ADON suggested that DOGT1 may catalyse the formation ofDON-3-O-glucoside. To test this hypothesis, first chemically synthesizedDON derivatives with the glucose moiety was attached either to the C3-or C15-hydroxyl group of DON. The two products were characterized withLC-MS/MS.

The DON-3-O-glucoside and DON-15-O-glucoside eluted at 12.43 min and12.68 min, respectively. The mass spectra of the glucosides showedcharacteristic differences in their fragmentation pattern (FIG. 5).While the DON-3-O-glucoside fragmented to an ion of 427.2 m/z under thegiven conditions, the same ion was not detected with DON-15-O-glucoside.The loss of 30 atomic mass units can be explained by the cleavage of the—CH₂OH group at C₆, which is prevented when the hydroxyl group isconjugated with glucose as in DON-15-O-glucoside. Further breakdown (MS)of the DON-glucosides in the linear ion trap showed an almost identicalfragmentation pattern as that of the [DON-H]-ion (295.3 m/z, notfragmented in Q2), confirming the presence of DON entities in thereaction products.

With these tools at hand it was possible to directly determine whichglucoside was formed in yeast cells. Yeast strain YZGA515, incapable ofconverting DON into 3-ADON due to a deletion of the yeastacetlytransferase gene AYT1, was transformed with both the DOGT1expression vector and a plasmid containing a gene encoding atrichothecene insensitive mutant ribosomal protein L3, the target oftrichothecenes, to increase DON tolerance of yeast cells. After theresulting strain had been incubated with high amounts of DON, reaching afinal concentration of 1000 ppm in the medium, the DON-metabolite wasextracted from the cells and identified as the expected3-O-glucopyranosyl-4-deoxynivalenol (FIG. 6A) by HPLC and massspectroscopy. FIG. 5 shows the fragmentation pattern of the synthesizedreference substances and the product peak from yeast expressing DOGT1.As expected, the metabolite was not present in the control strain,lacking DOGT1 activity.

In order to further verify substrate specificity, a GST-DOGT1 fusion wasconstructed. The GST-DOGT1 fusion gene was also expressed in yeast,where it conferred DON resistance like wild-type DOGT1. The gene productwas expressed in E. coli and affinity purified. The reaction productsgenerated in vitro during incubation of either the DOGT1 fusion-proteinor GST with UDP-[¹⁴C] glucose and DON were analyzed using TLC. A spotwith the same Rf value as the synthesized DON-glucoside was observed inthe reaction containing the GST-DOGT1 fusion protein but not in thecontrol.

Glucosylation of 4-deoxynivalenol severely reduces its toxicity. Toanalyze whether the 3-β-D-Glucopyranosyl-4-deoxynivalenol is lessphytotoxic than DON, a wheat germ extract coupled in vitrotranscription/translation system was used. As shown in FIG. 6B thepresence of 1 μM DON in the reaction inhibited protein translationsignificantly, leading to 36.8% reporter enzyme activity compared to thecontrol (100%). 5 μM of the toxin resulted in only 3.1% luminescenceremaining whereas 20 μM of the synthesized DON-3-glucoside onlyinhibited luciferase activity by 8%. These results proof thatglucosylation of DON is a detoxification process.

Overexpression of DOGT1 in Arabidopsis thaliana increases DONresistance. Transgenic A. thaliana constitutively expressing DOGT1 underthe control of a tandem 35S promoter were generated and the amount ofrecombinant protein in transformants determined by Western blottingutilizing the N-terminal c-Myc epitopetag. Seeds of the homozygous line1319/2, which was found to have a high DOGT1 expression level (FIG. 6C),and wild-type Col-0 as control were germinated on MS media containing5-30 ppm DON and grown for 5 weeks before the phenotype was analyzed.

The main phytotoxic effect observed was slow germination of wild-typeplants. Root formation did not occur at all, cotyledons developed butstarted to bleach before true leaves could be formed. After 3 weeks ofexposure to DON most of the wild-type seedlings had lost all green colorand ceased growth. DOGT1-overexpression lines showed also a clear delayin development. Compared to wild-type, germination occurred earlier,roots were formed, cotyledons did not bleach and true leaves appeared.The differences in DON sensitivity observed were most apparent on mediacontaining 15 ppm of toxin (FIG. 6D). Control transformants with emptyvector (containing solely the gentamycin resistance gene), and linesexpressing low amounts of DOGT1 were as sensitive as wild-type Col-0.

DISCUSSION

Biotechnological relevance. Fusarium diseases of wheat and barley are ofhigh economic significance for countries around the world. The UnitedStates for instance have been severely hit by Fusarium epidemics in thelast decade. Direct losses due to FHB for the US wheat producers havebeen estimated to average about $260 million annually and over theperiod 1998-2000 the combined economic losses for small grain cerealswere estimated at $2.7 billion (39). Deoxynivalenol contamination oflarge portions of the harvest can lead to high intake of the mycotoxin.Children are the population group most at risk to exceed the tolerabledaily intake level (TDI) for DON. In the problematic year 1998 80% of 1year old children in the Netherlands exceeded the TDI (7).

The high importance of Fusarium diseases justifies research on mycotoxininactivation, although resistance against the pathogen is most likely apolygenic trait in crop plants and toxin resistance just one of itscomponents. The production of trichothecenes is the only virulencefactor of the fungal pathogen which is experimentally confirmed so far,with the exception of pleiotropic mutations in MAP kinases affecting notexclusively virulence but also conidiation, perithecia formation,vegetative growth and mycotoxin production (40, 41). The present findingthat overexpression of DOGT1 leads to increased deoxynivalenolresistance in transgenic Arabidopsis opens new possibilities forbiotechnological approaches aiming to antagonize the fungal virulencefactor. The only result published so far relies on a Fusariumacetyltransferase (42) converting DON into 3-ADON, which is about 2-foldless toxic to laboratory animals and therefore no detoxification withinthe meaning of the present invention. The phenotype of transformed yeastand Arabidopsis, as well as data obtained using in vitro translationassays, demonstrate that the DON-glucoside exhibits reduced toxicity. Ingeneral, glucosylation converts reactive and toxic aglucones into stableand non-reactive storage forms, thereby limiting their interaction withother cellular components. The addition of a sugar blocks the reactivesite of the substance and consequently reduces toxicity for the plant.Such modifications are furthermore considered to provide access tomembrane-bound transporters opening exit pathways from the cytosol tofor example the cell wall or the vacuole (43).

Glucosylation of trichothecenes represents a detoxification process forplants. Yet, in the digestive tract of humans and animals themycotoxin-glucoconjugates could be easily hydrolyzed, regenerating thetoxin. The extent to which the DON-glucoside is transported to thevacuole or to the apoplast is currently unknown. Before one attempts touse transgenic plants overexpressing a DON-glucosyltransferase, itshould be investigated whether only the vacuolar glucoside or also toxinbound to cell wall material as “insoluble residue” is a source of“masked mycotoxin” (44), which escapes traditional analytical techniquesand may be of much higher toxicological relevance as currently assumed.The analytical tools and reference materials developed during this workis suitable to address these questions.

Inactivation of toxins by glucosylation seems to be a prominent naturalmechanism for resistance, enabling plants to cope with the enormousdiversity of toxic microbial metabolites they may encounter in nature.DOGT1 is one of 118 UGT genes in A. thaliana which are predicted toconjugate small molecules.

The expression of UGT genes in yeast is a valuable complementaryapproach to the widely used expression in E. coli, especially when therespective chemicals have targets in eukaryotes only. One problem isthat wild-type yeast cells are frequently “impermeable” to thesubstances of interest. Inactivating several ABC-transporters in thepresent host strain was a prerequisite for the approach to selectresistance conferring cDNAs. In the case of trichothecenes this allowedthe phenotypic detection of the activity of the expressed genes in asimple plate assay. In principle, this approach could be adopted formany other substances.

Gene evolution and substrate specificity. DOGT1 is located in a cluster(FIG. 1B) with 5 other members of family 73C on chromosome II(At2g36800) indicative for gene duplication by unequal recombinationevents (32). One could speculate that such a gene amplification eventprovides a selective advantage under toxin stress. DOGT1 and othermembers in the cluster have very similar protein sequences suggestingredundancy in enzymatic function. However, when testing the homologuesfor detoxification of trichothecenes, it was found that yeast expressingthe gene with the highest sequence similarity UGT73C6 was as sensitiveas wild-type to all toxins tested (as well as UGT73C3), while the secondclosest homologue UGT73C4 exhibited the same properties as DOGT1.Enzymatic activity towards different hydroxycoumarins has been shown forall 73C cluster members (45), indicating that the UGTs lackingDON-protective activity are not simply loss of function alleles. Thecluster is therefore an example that supports the hypothesis thatduplicated UGT genes can acquire new substrate specificities andfunctions during evolution.

Plant UDP-glucosyltransferases are structurally very similar in theircarboxy-terminal signature motif to mammalian UGTs which useUDP-glucuronic acid instead of UDP-glucose as donor substrate. Themammalian enzymes play a central role in metabolism and detoxificationof chemicals like carcinogens or hydrophobic drugs. Higher plant UGTshave been found to be involved in a parallel range of activities alsomodifying xenobiotic substances such as herbicides and other pesticides(46, 47). [One open question is whether these detoxification reactionsare side-activities of UGTs conjugating endogenous plant compounds.Although recent publications are in favor of the hypothesis that UGTsresponsible for the conversion of endogenous substrates may also accountfor the capacity of plants to detoxify xenobiotic substances (48) thisquestion will need further work to be solved.]

The finding that DOGT1 protects against DON but not NIV, which possessesone additional hydroxyl group at residue 4 (FIG. 1A), indicates that thevariation of the hydroxylation pattern of the trichothecene backbonemight be an important mechanism of the fungus to escape suchdetoxification reactions. It is unknown whether plants can alsoconjugate NIV, which has higher toxicity than DON in animal systems (5).The lack of activity against NIV stringlu argues against use of a singleDOGT1-like gene in transgenic plants, as NIV producing chemotypes of F.graminearum could get a selective advantage.

The construction of hybrid genes by shuffling the N- and C-terminal partof DOGT1 and UGT73C6 and their expression in yeast proved that theN-terminal 189 amino acids of DOGT1 are essential for its ability todetoxify DON, in agreement with the hypothesis that substrate bindingmay occur in a conserved hydrophobic area between amino acid 130 and 150(38). Comparison of two genes with protective effect and two geneswithout activity, but similar expression level, suggested that a lysine,which is present at amino acid position 136 in DOGT1 (and at thecorresponding position in UGT73C4) but replaced in the two UGT's unableto detoxify trichothecenes, could be important for DON detoxification.This hypothesis was falsified using site directed mutagenesis andexpression of the engineered proteins in yeast.

Information on specific domains or amino acids involved in acceptorbinding of DOGT1 may be potentially useful to search forglucosyltransferases capable to detoxify DON in crop plants like wheator maize. It should be considered though that attempts to assignfunctions to UGTs based exclusively on amino acid similarity are highlyerror prone, as shown in the case of the 73C cluster. Nevertheless, thefunctionality of candidate genes can be easily tested by expression inyeast. One possibility which can not be excluded is that glucosides oftrichothecenes are formed in crop plants by structurally fairlydissimilar UGTs. For instance, UGT84B1 was shown to exhibit the highestin vitro activity against the auxin indole-3-acetic acid (IAA) of all A.thaliana UGTs tested, yet this enzyme does not display the highestsequence similarity to the functional homologue in maize, the iaglu gene(49).

Regulation of gene expression. Genes with high sequence similarities toDOGT1 from tobacco and tomato have been shown to be induced by salicylicacid or wounding (34, 36, 37). The analysis of expression of DOGT1 and73C6 following SA and JA treatment showed that they responded weeklywith elevated mRNA levels to SA, JA and the ethylene precursor ACC.Inducibility of gene expression by SA, JA or ethylene is considered tobe indicative for a possible role of the upregulated gene product inplant stress or defense responses (50).

Using analysis of mRNA levels and a GUS-reporter construct it waspossble to show that DOGT1 transcription in wild-type A. thaliana isdevelopmentally regulated and is rapidly and significantly induced inresponse to DON exposure. Induction of expression by the mycotoxin hasalso been observed for other 73C cluster members, regardless of theirDON-protective activity. It would be interesting to clarify whether theinducibility is compound specific or a general phenomenon for proteinbiosynthesis inhibitors. The “ribotoxic stress response” is currentlyactively investigated by researchers in human cells (51), where themycotoxin induces the expression of cyclooxigenase-2, a key enzyme inthe synthesis of mediators of the inflammatory response, via MAP kinasemediated signaling. The DON inducible expression of DOGT1 is anattractive starting point for investigating whether similar mechanismsexist in plants.

With the present invention it was shown that the huge gene family ofglucosyltransferases, especially—as shown in the examples—UGTs plays animportant role in plant pathogen interaction by participating indetoxification of metabolites produced by microbes to increase theirvirulence on hosts. Similar to the large family of leucine-rich-repeattype resistance genes (52) involved in pathogen recognition, geneamplification and diversifying selection of UGTs could lead to a broadspectrum protection against fungal toxins. The selective pressure toescape such glucosylation reactions may be a driving force in evolutionof microbial biosynthetic reactions leading to a wide spectrum of toxinstructures, as observed for trichothecenes.

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Abbreviations:

FHB, Fusarium head blight; DON, deoxynivalenol; 15-ADON,15-acetyl-deoxynivalenol; NIV, nivalenol; UGT, UDP-glucosyltransferase;ABC, ATP-binding cassette; DOGT1, DON-glucosyltransferase; ORF, openreading frame; GUS, β-glucuronidase; SA, salicylic acid; JA, jasmonicacid; ACC, 1-aminocyclopropylcarbonic acid; TDI, tolerable daily intake;IAA, indole-3-acetic acid; DAG, days after germination.

1.-19. (canceled)
 20. A method comprising contacting a mycotoxin with aglucosyltransferase in the presence of an activated glucose.
 21. Themethod of claim 20, wherein the mycotoxin is glucosylated.
 22. Themethod of claim 20, wherein the mycotoxin is detoxified.
 23. The methodof claim 20, wherein the glucosyltransferase is UDP-glucosyltransferaseand the activated glucose is UDP-glucose.
 24. The method of claim 20,wherein the mycotoxin is a trichothecene.
 25. The method of claim 24,wherein the trichothecene has a free hydroxy-group at position C₃ andtwo hydrogen groups at C₄.
 26. The method of claim 20, wherein themycotoxin is deoxynivalenol, 15-acetyldeoxynivalenol, and/or15-Acetoxyscirpendiol.
 27. The method of claim 20, wherein the method isperformed in vivo using recombinant DNA technology and/orglucosyltransferase expression enhancing compounds.
 28. The method ofclaim 27, wherein the glucosyltransferase is expressed in a transgenicplant cell containing a recombinant glucosyltransferase and/or arecombinant regulating region for a glucosyltransferase.
 29. The methodof claim 27, wherein a mycotoxin-inducible promoter of aglucosyltransferase is used.
 30. The method of claim 20, wherein theglucosyltransferase is immobilised to a solid surface and a mycotoxincontaining solution or suspension is contacted with said immobilisedglucosyltransferase.
 31. The method of claim 20, wherein theglucosyltransferase is a UDP-glucosyltransferase corresponding tosubfamily 73C of Arabidopsis thaliana.
 32. The method of claim 31,wherein the glucosyltransferase is UDP-glucosyltransferase 73C5 and73C4.
 33. The method of claim 20, further defined as a method fordetoxification of trichothecenes in agriculture or beer production. 34.A recombinant cell being resistant to mycotoxins comprising aheterologous glucosyltransferase or an enhanced expression activity ofan endogeneous glucosyltransferase due to transgenic expressionregulating elements.
 35. The cell of claim 34, further defined as beingresistant to trichothecenes.
 36. The cell of claim 34, further definedas a plant cell or a yeast cell.
 37. The cell of claim 34, furthercomprising at least one ABC transporter which has reduced activity or isinactive.
 38. The cell of claim 37, wherein the at least one ABCtransporter has at least one pdr5 deletion.
 39. The cell of claim 34,further comprising three or more ABC transporters which have reducedactivity or are inactive.
 40. The cell of claim 39, wherein the three ormore ABC transporters have at least one pdr5 deletion.
 41. The cell ofclaim 37, further defined as comprising a deletion in pdr5, pdr10, snq2,yor1, pdr15, and/or ayt1.
 42. The cell of claim 34, further defined asresistant to Fusarium.
 43. A plant comprising the recombinant cell ofclaim
 34. 44. The plant of claim 43, further defined as resistant toFusarium.
 45. A method for detoxification of mycotoxins inmycotoxin-ontaining solutions or suspensions comprising obtaining a cellof claim 34 and adding it to the solution.
 46. The method of claim 45,further defined as a method for detoxification of trichothecenes inagriculture or beer production.